Biology 212
General Genetics
Spring 2007
Polymerase Chain Reaction Lab
Purpose: To familiarize you with the use of recombinant DNA techniques for characterizing genes, including the polymerase chain reaction (PCR) and agarose gel electrophoresis.
Background Reading: Hartl and Jones, Chap. 6 pp. 228-230.
Introduction: In addition to the Human Genome Project, which you will hear about in the lecture, there are major efforts underway to study the genes of many important model organisms such as mouse, zebrafish, and Drosophila. The mouse and Drosophila genome projects have been completed. Currently, scientists are making good progress on the zebrafish genome project.
The zebrafish, Danio rerio, is a member of the minnow family that originated in the Ganges River in east India and Burma. The zebrafish has been used in research since the 1970’s because they are small, easy to breed, and easy to raise in captivity. Since the zebrafish has become quite popular in genetic research, we will be learning the technique of polymerase chain reaction (PCR) using zebrafish clones. In an upcoming lab, we will also analyze the DNA sequences of some zebrafish clones.
In one important phase of the zebrafish genome project, cDNA copies of zebrafish expressed genes have been cloned into a bacterial plasmid vector. Complementary DNAs are DNA sequences copied from the mRNAs of expressed genes. In higher eukaryotes, cDNAs lack the intron sequences present in many genes and are thus smaller and easier to work with than the chromosomal genes. The cDNA clones you will work with were genetically engineered so they could be inserted between the two Xho I sites in the cloning vector, pme18s-fl3, see next page. The vector pme18s-fl3 contains the gene for ampicillin resistance (ampR); bacteria containing the plasmid are selected in media containing ampicillin. Unlike most other vectors you might work with, pme18s-fl3 lacks a marker gene which can be disrupted by cloning.
Each person will be assigned a different zebrafish cDNA clone. You will be characterizing the individual zebrafish cDNAs in two ways. You will carry out PCR to determine the size of the zebrafish cDNA insert in the plasmid vector. Later, you will use a computer to analyze the zebrafish cDNA sequence to identify the protein it encodes and locate other features (Bioinformatics lab).
Week 1: Polymerase Chain Reaction (Set up takes about 10min.; PCR reactions take about 2-3 hours, but are carried out automatically by the thermocycler; instructor will remove the tubes later to be stored in the refrigerator until the next lab).
Background: The polymerase chain reaction is a method widely used for DNA testing, forensics etc. A very small amount of biological material (a few cells, a single hair) can serve as the source of DNA. In PCR:
· Small oligonucleotide primers bind to the two denatured strands of the DNA
· Primers are used to initiate the synthesis of new copies of the DNA.
· The reaction also requires:
o heat-stable DNA polymerase (synthesizes DNA)
o buffer (maintain optimal pH of solution)
o magnesium (Mg2+) ion (stabilizes enzyme in presence of negatively charged DNA)
o deoxynucleotides (the building blocks of DNA)
· After multiple cycles of denaturation, priming and DNA synthesis, enough DNA is produced to visualize by agarose gel electrophoresis.
· Agarose gel electrophoresis separates DNAs based on their sizes. The sizes of the DNA products of PCR can be determined by comparison to size marker, in a manner similar to sizing restriction fragments.
Procedure:
1. Practice using the micropipets. There will be a micropipette work station in the front of the lab set up where you can learn how to adjust the volume settings on micropipets and practice using them.
2. Sign up for a time to take the pipeting practical and set up the reactions. Some additional options for setting up the lab will be available on Friday afternoon.
3. Sign out a sample for an unknown zebrafish cDNA on the sign out sheet.
4. Label one thin-walled 0.2 ml tubes on the cap with your cDNA clone #.
5. The reactions must be set up with the special filter tips in the work areas provided to try to minimize DNA contamination of samples.
component / zebrafish cDNA clone #Master mix*: PCR buffer, MgCl2, deoxynucleotide triphosphates, forward primer, reverse primer, Taqurate Taq Polymerase / 23 ml
zebrafish plasmid DNA 2.5 ng/ml / 2 ml
* Final concentrations of components in master mix: 50 mM Tris-HCl (pH 8.3, 22º C), 50 mM KCl, 200 uM each dNTP, 2 mM MgCl2, 0.1 unit of Taqurate polymerase
6. Bring the samples to the instructor to be placed in the thermocycler. She (or a student she designates) will set up a control tube (no template control, consisting of 23 ul of Master mix + 2 ul sterile ddH2O), for each run. There will be a demonstration of the thermocycler, but reactions will be started together.
The BioRad Thermocycler will be programmed as follows:
The samples are first heated at 94°C for 2 min.
The cycle conditions are as follows:
94° C for 40 sec.
55° C for 40 sec.
72° C for 60 sec.
Repeat for 40 cycles
7. The instructor will store the samples at 4°C until the next lab period, when we will carry out the agarose gel electrophoresis.
Week 2: Agarose Gel Electrophoresis (Plan on about half an hour to cast the gel, 10 min. to load the samples and about an hour to run the gel.)
Overview:
· Setting up the gels takes about 30 min. and will be done prior to the regular class start time.
Extra credit option: (3 points)
Tues. 9:15 am Room 205
o Sign up to assist instructor with casting the gels.
· You should determine how many individual’s samples can be run on a particular gel and plan the order of the samples on the gels before you start. Since we have limited equipment, you will need to coordinate with your lab partner and perhaps with others in the lab to share gels and equipment.
· Before you leave today, particularly if you need to go before your gel has finished, you should leave the instructor with a diagram of the lanes and names of the persons who shared the gel, on the index card provided.
Preparation of gels: (done in advance of the lab)
1. Tape the ends of the casting tray or position tray sideways to create end walls, as demonstrated by the instructor. Position a comb near one end of the tray to create sample wells.
2. For small gels, weigh out 0.5 g of agarose on the balance and transfer to a 125 or 250 ml flask. Measure out 50 ml of 1x TBE electrophoresis buffer in a graduated cylinder and add to the flask. For larger gels, weigh out 1.0 g of agarose, transfer to a 250 ml flask and add 100 ml of 1x TBE buffer.
3. Microwave the flask for 2-3 min. to dissolve the agarose. Cool the agarose slightly.
4. Wearing gloves, add 1 ml of 10 mg/ml ethidium bromide (carcinogen) to the flask.
5. Carefully pour the gel solution into the casting tray. Allow to cool for 20 min. or so until the gel becomes opaque.
6. While the gel is cooling, design your electrophoresis experiment and prepare your samples for electrophoresis.
DESIGN YOUR EXPERIMENT
7. a. The PCR reactions from last week will be available in the front of the room
b. PREPARE SAMPLES FOR ELECTROPHORESIS
· Combine 7 ml of your PCR sample with 3 ml of a 5x load solution that is more dense than the electrophoresis buffer (ours contains glycerol mixed with a dye, bromophenol blue).
· To accurately size your unknown DNA fragments, you should include size markers in separate lanes, both lambda DNA digested with HindIII and the 100 bp ladder. To conserve on standard DNAs, they have already been made up for you with DNA and load solution; you will need to load 10 ml of each type to have an appropriate amount of DNA to visualize on the gel.
· Also include a positive PCR control lane. This is a sample of a PCR reaction already known to produce a product. If your own PCR reaction doesn’t turn out, you should analyze the results from this lane instead. This sample is also premixed with loading dye, so load 10 ml into the appropriate lane.
Suggested sample volumes: DNA volume sterile water load solution
Your PCR reaction 7 ml ------3 ml
Positive control PCR reaction 10 ml sample
Lambda/Hind III size marker ------10 ml sample
100 bp ladder ------10 ml sample
8. Plan out the contents of your sample wells for electrophoresis. Use a ten or twenty microliter micropipet to mix together the PCR samples and load solution in separate microfuge tubes, then load the entire contents in the appropriate wells.
9. Determine the samples that you and other lab partners need to run and make sure there will be enough spaces in the gel.
For example, if your gel has 8 wells:
Lane# Contents
Lane 1: Lambda/Hind III
Lane 2: 100 bp ladder
Lane 3: empty
Lane 4: PCR reaction person #1
Lane 5: PCR reaction person #2
Lane 6: PCR reaction person #3
Lane 7: PCR reaction person #4
Lane 8: Positive PCR control
10. When your gel is solidified, put gloves on to remove the tape from the ends, place the gel in the electrophoresis chamber and cover the gel with 1x TBE electrophoresis buffer.
11. Use a micropipet to load your samples into the designated wells. Be sure to write down the order of your samples in your notes.
12. Connect the leads of the gel apparatus to the power supply so that the DNA will migrate toward the positive pole (red electrode) and away from the negative pole (black electrode).
13. Carry out the electrophoresis at 100 V for 45 min. to 1 hour or until the dye front has moved about 1/2 the way down the gel. Turn off the power supply and disconnect the leads.
14. Remove the gel from the tank (wear gloves!) and view the gel on the transilluminator. Your instructor will help you photograph your gel. Used ethidium bromide stained gels should be saved in the designated waste container. When finished, rinse out the electrophoresis units with distilled water and set on the counter to dry.
15. Most gels will be photographed using a DNA imaging system. On an index card in your work area, please provide instructor with information on your gel and who the lab partners were. A printout of your results will be provided to you.
16. If you want a digital copy of your gel, also provide the instructor with an email address, so JPEG files of your results can be forwarded. The image files of the gels are rather large. It is better to analyze a print-out that is roughly the size of the original gel. What I typically do is create a word file and insert the picture into the word file where it can be resized and labeled.
Laboratory Assignments:
1. A 10 point lab practical on pipeting and laboratory calculations, given in lab on Tuesday April 10 or by appointment on Thursday morning or Friday afternoon; The practical must be taken before you begin the lab.
· Be able to use p20 and p200 micropipets to measure and transfer liquids. Understand what volumes of liquids these are appropriate to measure.
· Be able to interconvert microliter and milliliter units. (1 ml = 1000 μl)
· For samples with a given concentration, be able to calculate a given volume containing a certain mass of DNA.
2. A 10 point quiz will be given in lab on Tuesday April 17 on the theory and procedures used in the Polymerase Chain Reaction. Study this handout, class and lab notes and assigned text pages.
3. Each person must characterize the cDNA they were assigned. Complete the following worksheet (10 points) on or before Friday April 27.
PCR/Gel Electrophoresis Worksheet (10 pts.) Name______
Section#______
Zebrafish cDNA#______
1. a. Did your PCR reaction work? Could you see your PCR product on the agarose gel?
b. If your PCR reaction did not work, what would you do differently next time?
2. Attach a copy of the picture of the gel you ran. This should be the same image you use in the measurements below. Label the standard lanes, the lane with your PCR reaction, and the positive PCR control.
3. Gel Standard Curve. Use a centimeter ruler to measure the distance the size markers traveled. Measure from the bottom of the well to the middle of the DNA fragment.
a. Complete the table below:
Lambda/HindIII / Lambda/
HindIII / 100 bp
ladder / 100 bp ladder
distance (mm) / bp / distance(mm) / bp
23,000 / 1,500
9,400 / 1,200
6,600 / 1,000
4,400 (faint) / 900
2,200 / 800
2,000 / 700
560 / 600
125 (not seen) / 500
400
300
200
100
b. On the semilog graph paper provided, construct a standard curve using the size markers. For each of the fragments from the Lambda/Hind III lane and the 100 bp ladder lane, plot the distance migrated in mm (X-axis) against the size in base pairs (Y-axis). Be sure to correctly label the axes.
4. Use a centimeter ruler to measure the distance the major PCR product traveled. Use the standard curve to determine the size of the cDNA insert for your zebrafish clone (or the positive PCR control, if you had no PCR product) and complete the table.
Distance traveled (mm) / Predicted cDNA size (bp)8